Aging is associated with progressive immune dysfunction, resulting in blunted effector responses and increased susceptibility to infectious disease and cancers. A significant contributor to this decreased responsiveness is the impaired functionality of the most potent Ag-presenting cells, the dendritic cells (DCs). A key aspect of DC function is their capacity to ?cross-present? cell-associated Ag to CD8+T cells, a process that is required to cross-prime CD8+T cell responses against tumors or intra-cellular pathogens. However, the effect of aging on the acquisition, processing, and presentation of cell-associated Ags by primary DCs has barely been studied. We recently uncovered that aging significantly reduced the cross-presenting and cross-priming capacity of the DC subsets the most efficient at carrying out this important task (CD8?DCs and merocytic DCs). Aged DCs expressed normal MHC class I levels and were able to prime CD8+T cells to soluble peptides, but they were strikingly impaired in terms of phagocytosis of cell-associated materials. Mechanistically, the bio-energetic status, notably mitochondrial activity and membrane potential (??m), has been shown to regulate DC phagocytic capacity. Mitochondrial function was profoundly affected in murine CD8? or mcDCs, as shown by lower ??m, reduced ATP turnover and coupling efficiency, decreased baseline oxidative phosphorylation, and greater proton leak and ROS production. Old macaque CD1c+ DCs displayed similar defects, demonstrating dysfunction across species. Pharmacologic manipulation of young DCs to mimic the aged metabolic phenotype significantly impeded their phagocytic and cross-priming capacity, but in vitro scavenging of ROS by NAC significantly reversed the cross-presentation defect of aged! DCs. Our inter-related hypotheses are: (1) age-related changes in mitochondrial activity diminish the phagocytic and cross-presenting activity of murine DCs; and (2) improving mitochondrial activity will boost DC functionality and their potential as anti-tumor therapeutics in the elderly. These hypotheses will be tested in the following 3 aims. Aim 1 will identify the mechanisms underlying decreased mitochondrial fitness in aged murine DCs. We will dissect the effect of aging on DC use of, and dependence on, specific carbon sources and metabolic pathways to meet their energy needs. Moreover, we will assess whether the observed metabolic changes result from defects in the DC precursor, the aged environment in which they differentiate, or a combination of both. Aim 2 will determine whether improving mitochondrial activity of aged murine DCs increases their capacity to induce more vigorous T cell responses to cell-associated Ags in vitro, while Aim 3 will study the effect of improving mitochondrial function on their capacity to induce anti-tumor responses using well-established in vivo models of cross-presentation. !